Adolescents - The Nutrition Society of Sri Lanka

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May 16, 2007 - Medicine, University of Kelaniya, Ragama, Sri Lanka and 3Department of Paediatrics, Baylor College of Medicine, Houston, TX, USA.
European Journal of Clinical Nutrition (2008) 62, 856–865

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ORIGINAL ARTICLE

The efficacy of micronutrient supplementation in reducing the prevalence of anaemia and deficiencies of zinc and iron among adolescents in Sri Lanka M Hettiarachchi1, C Liyanage1, R Wickremasinghe2, DC Hilmers3 and SA Abrams3 1 Nuclear Medicine Unit, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka; 2Department of Community Medicine, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka and 3Department of Paediatrics, Baylor College of Medicine, Houston, TX, USA

Objective: To determine the effectiveness of combined iron and zinc over the iron or zinc-only supplementation in correcting deficiency and possible interactive effects in a group of adolescent school children. Subjects and methods: Schoolchildren (n ¼ 821) of 12–16 years of age were randomized into four groups and supplemented with iron (50 mg/day), zinc (14 mg/day), iron þ zinc or placebo capsules 5 days per week for 24 weeks. Anthropometry, and haemoglobin (Hb), serum zinc (SZn) and serum ferritin (SF) concentrations were determined before and after the intervention. Results: There were no significant effects between-groups in their weight, height and Hb concentrations with the intervention when compared with the placebo group. Iron-only and combination-supplemented groups had reached mean SF concentrations of 55.1 mg/l with no difference between them (P ¼ 0.99). The zinc-only group had a mean change of 4.3 mmol//l whereas the combine-supplemented group had a mean change of 4.0 mmol/l (P ¼ 0.82). The prevalence of anaemia was found to be 70.3% in the iron group at baseline; this was reduced to 14.5% after the supplementation. In the combinesupplemented group anaemia, prevalence was reduced from 64.8 to 19.3%. Conclusions: Zinc alone or in combination with iron has not shown a significant improvement in growth in adolescence. Severe and moderate forms of anaemia were successfully treated in children who received iron supplementation. Initial high prevalence of low SZn and iron stores was significantly improved with micronutrient supplementation.

European Journal of Clinical Nutrition (2008) 62, 856–865; doi:10.1038/sj.ejcn.1602791; published online 16 May 2007 Keywords: adolescence; randomized controlled trial; iron supplementation; zinc supplementation; iron and zinc interactions; school children

Introduction A high prevalence of micronutrient deficiencies presents a major obstacle to socio-economic development in developing Correspondence: Dr M Hettiarachchi, Nuclear Medicine Unit, Faculty of Medicine, University of Ruhuna, PO Box 70, Galle, Sri Lanka. E-mail: [email protected] Guarantor: C Liyanage. Contributors: MH was responsible for the research project and involved in all aspects; designing, sample selection, data collection, results and statistical analysis, paper writing and so on. CL was responsible for the research project and involved in all aspects; designing, supervision of sample selection and data collection, results and statistical analysis, paper writing and editing. RW was responsible for the research project and involved in designing of the sample size, statistical analysis and editing the paper. DCH Involved in project designing, results analysis and editing the paper. SAA was involved in project designing, results analysis and editing the paper. Received 3 May 2006; revised 20 March 2007; accepted 11 April 2007; published online 16 May 2007

countries with an immense impact on the health of the population, as well as its productivity (Preventing micronutrient malnutrition, 1997). Populations in South Asia, including Sri Lanka, have the lowest daily intake of zinc in the world; and 44% in Sri Lanka are at risk for zinc deficiency (Wuehler et al., 2005). Diets that provide inadequate amounts of zinc are likely to provide inadequate amounts of iron as well, because meats and fish are the best sources of both nutrients (Allen, 1998). Supplementation has been identified as the best option where the prevalence of a micronutrient deficiency in a population is high, and especially if the requirement for a nutrient is difficult to achieve through the normal diets in well-defined target groups. Combining multiple micronutrients in a single delivery system has been suggested as a cost-effective strategy to ameliorate this problem (Nilson and Piza, 1998). However, it

The efficacy of micronutrient supplementation M Hettiarachchi et al

857 has been reported that iron and zinc interact during absorption and possibly during metabolism because they have chemically similar absorption and transport mechanisms (Sandstrom, 2001). This becomes particularly relevant in populations with poor iron and zinc status. New evidence based on cell culture studies has shown that iron may inhibit zinc absorption in some cells at very high ratios of iron to zinc, but not vice versa (Kordas and Stoltzfus, 2004). It has been suggested that adolescence may be an optimal period in which to deliver micronutrient supplements to build stores (Lynch, 2000). The physiological demand for iron is high during adolescence because of the expansion of the blood volume associated with the adolescent growth spurt and the onset of menstruation (Dallman, 1992). It is also a time when supervised supplementation may be possible, in school-based programmes, for example. However, evidence of antagonism from studies of low ratios of iron to zinc is needed to assess any biochemical and functional effects of dual supplementation. Assessing the effect of single zinc or iron supplementation on the biochemical indicator of the other (that is, zinc on iron and iron on zinc) may help shed light on whether adverse effects are associated with supplementation. With this background in mind, we investigated the efficacy of iron and zinc supplementation in improving anthropometry, and haemoglobin (Hb), serum zinc (SZn) and iron stores (serum ferritin, SF) in school children aged 12–16 years as part of a larger nutritional intervention programme aimed at improving micronutrient status among adolescents in Sri Lanka. Furthermore, the effect of supplementation on pubertal growth was also investigated. This investigation was carried out in the Galle District of Sri Lanka, where iron and zinc deficiency has been documented to be highly prevalent in earlier studies (Hettiarachchi et al., 2006).

Subjects and methods Children 12–16 years of age attending schools in the Galle District constituted the study population. Three schools (only girls school and two co-ed schools) were randomly selected. The selected schools had urban and rural children in equal proportions. The principals of the schools and the class teachers were briefed about the research project and then the subjects and their parents were informed by a letter, which described in detail the procedures involved in the study. Children who presented written consent from their parents were enrolled in the study after explaining in detail the research programme to them. The study was approved by the Ethical Review Committee of the Faculty of Medicine, University of Ruhuna, Sri Lanka.

Screening The selected sample of children was subjected to a comprehensive physical examination, including measurements of height and body weight using standardized scales. A sample of venous blood (3 ml) was drawn for the initial assessment of micronutrient status. An aliquot of 200 ml of whole blood was pipetted onto a filter paper for measurement of Hb, and rest of the blood was collected in acid-washed centrifuge tubes, and transferred to the laboratory, where serum was separated by centrifugation and kept frozen at 201C until SZn and SF analyses were carried out. The dried filter paper was placed in a test tube containing Drabkin’s solution for the Hb measurement on the following day.

Sample size On the basis of the estimated prevalence of anaemia (defined as Hbo 120.0 g/l) in this age group in Sri Lanka (Mudalige and Nestel, 1996), the sample size was calculated to be 180 per group to demonstrate a 15% reduction in the prevalence of anaemia with an a error of 5% and a b error of 10%. The calculated sample size was inflated to 200 per group assuming a 10–12% dropout rate during the follow-up period.

Enrolment Children with Hb level X80 g/l were eligible for the study. Others were referred for further investigations and treatment. Further, children suffering from acute or chronic diseases, inflammatory conditions, giving a history of any drug consumption other than paracetamol or antihistamines for minor ailments, currently consuming nutrient supplements or having donated blood or received a blood transfusion within the last 4 months were excluded from the study. Of the children screened, 821 met the inclusion criteria and participated in the study. A letter was given to their family physicians requesting notification if they wished to prescribe additional micronutrient supplements during the study period. All the study subjects were treated for parasites by giving mebendazole (500 mg) as a single oral dose (mass-treatment) approximately 2 weeks before the start of the study.

Drug supplementation Subjects were randomized into one of four groups where randomization was stratified by classroom using a doubleblind approach. Four groups of children were supplemented with two capsules per day containing either iron (50 mg/day) in the form of ferrous fumarate, zinc (14 mg/day) in the form of zinc sulphate, combined (iron þ zinc) or placebo made of anhydrous lactose. The composition of the supplement was based on the daily recommended allowance (RDA) of the World Health Organization (1992) for zinc, and global guidelines for iron supplementation of the International European Journal of Clinical Nutrition

The efficacy of micronutrient supplementation M Hettiarachchi et al

858 Anaemia Consultative Group/World Health Organization/ UNICEF (Stolzfus and Dreyfuss, 1998). The subjects received capsules daily on school days for a period of 24 weeks from their class teacher. The capsules were consumed each morning at the time daily attendance was taken. Teachers were instructed to ensure that their students consumed the capsules. Capsules were provided to the class teachers by the investigators every 2 weeks; the teachers were asked to sign a checklist when the doses were given. These checklists were randomly checked for accuracy and compliance. The final assessment was carried out 1 week after the end of the intervention period. During this assessment, anthropometric measurements were taken, a medical examination was carried out and 3 ml of venous blood was drawn for determination of Hb, SZn and SF concentrations.

(SZn and SF) were normally distributed. Because the distributions of Hb, SZn and SF values were skewed, they were log-transformed in all calculations. For presentation, these variables were transformed back to the original scale. Paired-t-tests were used to study within-group treatment effects. Between group treatment effects (baseline compared with post-intervention) in anthropometric indices and concentrations of biochemical parameters were analysed using repeated measures design. Baseline values of respective parameters were also included in the analysis as a covariant for between-subject factors to correct for their possible confounding influence on the change in weight, height, Hb, SZn and SF. The w2 test was used to compare prevalences of deficiencies between groups. All analyses were carried out using SPSS version 10.0 for Windows (SPSS Inc., Chicago, IL, USA).

Biochemical analysis Hb concentration was measured photometrically using the cyanmethaemoglobin method at the Nutrition Research Laboratory, Faculty of Medicine, Galle. SF was measured by immunoradiometric assay at the Nuclear Medicine Unit, Faculty of Medicine, Galle using reagents supplied by NETRIA, England. SZn was analysed at the Industrial Technology Institute (ITI), Colombo by flame atomic absorption spectrophotometry (f-AAS).

Results In the sample (821) of children studied, the mean age, baseline status of growth was categorized according to their group allocation (Table 1). There was no difference in the prevalence of stunting and underweight in the four groups. The iron-only group had a higher prevalence of anaemia and iron deficiency. Similarly, zinc deficiency was higher in the zinc-supplemented groups (zinc only and combination). At baseline, 35.1% (n ¼ 288) of subjects had low Hb and low SF concentrations. A total of 30.2% (n ¼ 248) had low SF and low SZn and 20.2% (n ¼ 166) had low concentrations of all three indices. Of the children recruited, 774 completed the 24-week study protocol. A total of 47 (5.7%) children were dropped for various reasons: withdrawal from the study (n ¼ 23), refusal to give blood after supplementation (n ¼ 7), and absence on the day of the post-supplementation blood

Statistical analysis The 1978 CDC/WHO growth reference curves were used from Epi-info version 3.0. (2003) to generate values for z scores of weight-for-age (WAZ), and z scores of height-for-age (HAZ). Body weight and height less than 2 s.d. of reference standards were considered as cutoff points for undernutrition. A one-sample Kolmogorov–Smirnov test was used to determine whether the anthropometry (weight, height and body mass index (BMI)), Hb and serum biochemical markers

Table 1 Anthropometric indices at baseline and at the end of the supplementation studya Parameter

n Mean age (years) Underweightb Stuntedc Weight (kg) Height (cm) BMI WAZ HAZ

Iron

Zinc

Combine

Placebo

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

202 14.2 33.7 19.0 39.53 (8.17) 151.79 (8.09) 16.96 (2.54) 1.68 (1.24) 1.27 (0.89)

193

213 13.3 39.0 18.8 37.11 (8.42) 149.86 (9.61) 16.30 (2.45) 1.69 (1.24) 1.09 (1.04)

201

216 12.9 35.7 17.8 36.34 (8.04) 149.41 (8.14) 16.16 (2.38) 1.58 (1.23) 1.05 (0.98)

199

190 13.6 41.1 16.8 37.23 (8.77) 150.82 (9.14) 16.19 (2.50) 1.74 (1.25) 1.10 (1.07)

181

39.95 152.22 17.25 1.83 1.41

(7.92) (8.09) (2.79) (1.27) (0.90)

38.18 150.98 16.58 1.72 1.14

(8.48) (9.36) (2.47) (1.27) (0.99)

36.63 150.14 16.96 1.69 1.24

(7.78) (8.03) (2.59) (1.26) (0.98)

37.60 151.40 16.23 1.99 1.30

(8.83) (9.23) (2.51) (1.35) (1.02)

Abbreviations: BMI, body mass index; HAZ, height-for-age z score; WAZ, weight-for-age z score. a Results are expressed as mean (s.d.). None of these parameters was significant when compared against placebo (repeated measures ANOVA) at baseline and after 6 months of supplementation. b Defined as WAZp2.00. c Defined as HAZp2.00.

European Journal of Clinical Nutrition

The efficacy of micronutrient supplementation M Hettiarachchi et al

859 collection (n ¼ 17). Baseline parameters of the subjects who entered the final analysis were not significantly different from that of the subjects who did not enter the final analysis (data not shown). On the basis of compliance records, 58% of the subjects received possible 120 doses, 32% received 110–119 doses and only 2% of subjects received less than 100 doses. The mean number of capsules consumed by groups was not significantly different (P ¼ 0.36).

Anthropometric indices Weight and height at baseline were not significantly different between treatment groups. Following 24 weeks of supplementation, all four groups had a significant withingroup improvement (Po0.001) in anthropometry (that is, weight, height, BMI, WAZ and HAZ) from their respective baseline values. However, there were no significant treatment effect between-groups in their weight and height improvement (Table 1). During the intervention, the BMI of the supplemented groups significantly increased from their respective baseline status when compared to the placebo group (0.32 in supplemented, 0.04 in placebo; Po0.001). The combination group had lower mean WAZ and HAZ at baseline than the other three groups, but this difference was not significant (P ¼ 0.08). Changes in the WAZ and HAZ were positively correlated with the age of the subjects (Po0.001) and negatively correlated with their respective values at baseline (Po0.05). The changes in z scores were not significant when compared with the placebo group (Table 1).

Biochemical changes Despite the randomized allocation of children to treatment groups, Hb, SF and SZn concentrations in groups at baseline were significantly different (Po0.001). Mean Hb concentration in the placebo group (121.5 g/l) was shown to be significantly higher than in the other three groups (Po0.001). The mean SF in the placebo group (34.4 mg/l) was significantly higher than in iron-supplemented groups

(16.5 mg/l in iron- and 22.7 mg/l in the combine-supplemented group; Po0.001) but not in the zinc-only supplemented group (mean concentration of 31.1 mg/l, P ¼ 0.56). Baseline SZn concentration in the iron group (10.8 mmol/l) was significantly different from the zinc-supplemented groups (Po0.001) but not with the placebo group (10.2 mmol/l, P ¼ 0.99). Further, there was no difference in SZn between zinc alone and the combination groups (mean levels of 7.3 and 7.9 mmol/l respectively, P ¼ 0.27). These initial differences were considered during analysis (see Statistical analysis). Micronutrient supplementation resulted in significant within-group increases in Hb, SF and SZn in all four groups (Po0.001) except in the case of SF in the placebo group (Table 2). The iron-only group had mean Hb increase of 18.2 g/l and the combination-supplemented group had and Hb increase of 11.0 g/l during the intervention. The prevalence of anaemia was 70.3% in the iron group at baseline; this was reduced to 14.5% after supplementation. In the combination-supplemented group, the prevalence of anaemia was reduced from 64.8 to 19.3% and, in the zincsupplemented group, the prevalence reduced from 52.6 to 26.1%. In the placebo group, there was a slight increase from 38.9 to 43.1% (Figure 1). The difference observed in the SF concentration of the iron- and combination-supplemented groups at baseline over the other two groups was significantly reversed with the intervention (Table 2). Both groups were similar and had mean concentrations of 55.1 mg/l (P ¼ 0.99). There was no difference in SF between the zinc and the placebo groups (mean concentration of 34.5 and 34.1 mg/l respectively; P ¼ 0.07) during this period. Figure 1 illustrates that iron stores had improved in the iron-supplemented groups. The baseline prevalences of iron deficiency of 80.5% had reduced to 7.3% in the iron-only group and 65.3% of iron deficiency in the combination-supplemented group was reduced to 6.5%. The zinc and placebo groups had a prevalence of iron deficiency of more than 35.0% (37.31% in zinc; 39.2% in placebo) even after the intervention.

Table 2 Biochemical indices at baseline and at the end of the supplementation studya Parameter

N Hb (g/l) SZn(mmol/l) SF (mg/l)

Iron

Zinc

Combine

Placebo

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

202 116.05 (1.14) 10.79 (1.14) 16.50 (1.14)

193 130.13 (1.08) 11.03 (1.27) 55.09 (1.54)c

213 117.92 (1.12) 7.32 (1.53) 31.12 (1.04)

201 125.99 (1.10) 11.98 (1.24)b 34.54 (1.69)

216 115.58 (1.09) 7.92 (1.50) 22.73 (1.94)

199 127.22 (1.08) 11.99 (1.24)b 55.07 (1.52)c

190 121.51 (1.21) 10.18 (1.49) 34.43 (1.88)

181 119.81 (1.23) 10.80 (1.21) 34.06 (1.81)

Abbreviations: Hb, haemoglobin; SF, serum ferritin; SZn, serum zinc. a Results are expressed as mean (s.d.). Hb concentration in the placebo group at baseline was significantly higher than the other three groups (ANOVA; Po0.001). Baseline SZn concentrations in the iron group was significantly different from zinc-supplemented groups (Po0.001) but not with the placebo group (P ¼ 0.99). There was no difference in SZn between zinc and combined groups (P ¼ 0.27). The mean SF in placebo group was significantly higher than in iron-supplemented groups (iron and combine-supplemented; Po0.001) and, no difference with the zinc group (P ¼ 0.56). b Zinc-supplemented groups (zinc only and combine-supplemented) had significant improvement in SZn concentrations (repeated measures ANOVA; Po0.001). c Iron-supplemented groups (iron-only and combine-supplemented) had significant improvement in iron stores (repeated measures ANOVA; Po0.001).

European Journal of Clinical Nutrition

The efficacy of micronutrient supplementation M Hettiarachchi et al

860 deficiency prevalence during the intervention (42.6% at baseline to 35.8% in the iron-only group and from 40.5 to 33.1% in the placebo group). The effect of the intervention was more marked among those who were deficient in the respective micronutrients and anaemic at baseline (Figure 2). Anaemic children at baseline had a mean change of þ 12.7 g/l in Hb with the intervention whereas non-anaemic had mean change of þ 4.2 g/l (Po0.001). SF showed a similar pattern of improvement where the mean changes were þ 23.6 mg/l in SFo30 and þ 11.0 mg/l in SF sufficient groups (Po0.05). However, in the iron-supplemented groups, SF sufficient children had a better improvement in SF than deficient children (Figure 2). Iron-sufficient children in non-ironsupplemented groups had a reduction in mean values but iron-deficient children had a significant positive improvement after follow-up (Po0.05). Zinc-deficient children in all four groups had positive and higher mean changes in SZn concentrations (overall value of þ 3.8 mmol/l) as compared to zinc-sufficient children ( þ 0.5 mmol/l mean change, Po0.001).

Effect on pubertal growth and micronutrient status As daily iron and zinc requirements are different between adolescent males and females and iron requirements vary between pre menarcheal and post-menarcheal females, we compared the treatment effect in these subcategories separately. There were 327 males enrolled, of whom, 321 completed the trial. A total of 217 premenarcheal females were enrolled and 206 were available for the final assessment. Three premenarcheal females did not complete the study and another eight females who attained menarche during the intervention period, were excluded in the analysis. There were 277 post-menarcheal females of which 30 were dropped out during follow-up.

Figure 1 Prevalence of anaemia, zinc and iron deficiencies at the beginning and at the end of the intervention.

Similar to the improvement of iron stores in the ironsupplemented groups, SZn levels were improved in the zincsupplemented groups (Table 2). The zinc-only group had a mean change of 4.3 mmol//l whereas the combine-supplemented group had a mean change of 4.0 mmol/l (P ¼ 0.82). The zinc-only group had baseline SZn deficiency prevalence of 73.2% which was reduced to 25.3% while the prevalence in the combine-supplemented group fell from 62.0% at baseline to 17.8% after the intervention (Figure 1). Non-zincsupplemented groups showed a slight reduction in the SZn European Journal of Clinical Nutrition

Males There were no significant differences between groups in all anthropometric indices at baseline. Zinc alone supplementation had significantly improved (P ¼ 0.05) height of males (Table 3). The placebo group had a significantly higher Hb concentration (Po0.05) at baseline. SF concentrations of placebo and zinc-only groups at baseline were significantly different (Po0.001) from that of the other two groups. SZn levels of iron-only and placebo groups were different from other two groups (Po0.05) at baseline. Mean SZn concentration in zinc-only and combinesupplemented subgroups showed a significant improvement with intervention (P ¼ 0.05) when compared with the placebo group (Table 4). Other biochemical parameters (that is, Hb and SF) also showed a positive effect with micronutrient supplementation, but the improvements were not significant.

The efficacy of micronutrient supplementation M Hettiarachchi et al

861 Post-menarcheal females As among males, there was no difference in anthropometric status between groups at baseline in post-menarcheal females (Table 3). The mean Hb (107.8 g/l) and SF (16.1 mg/l) concentrations were significantly lower in the iron-only group (Po0.001) compared to the other three groups. SZn concentrations were significantly (Po0.05) lower in zinc-supplemented groups (i.e., zinc-only 6.4 mmol/l and combination supplemented 7.6 mmol/l) when compared with the other two groups. There was no significant treatment effect on weight, height and BMI of postmenarcheal females (Table 3). WAZ significantly improved among the combine-supplemented group (P ¼ 0.05) and in both zinc-and combine-supplemented groups there was a positive impact on HAZ (P ¼ 0.03). All three micronutrient supplemented groups had improved Hb concentration with the intervention (Po0.001) when compared with control group (Table 4). SF concentration was significantly improved in iron-supplemented groups (P ¼ 0.03) whereas SZn concentration was significantly improved (P ¼ 0.03) only in the zinc-only supplemented group.

Premenarcheal females There was no difference between groups at baseline in anthropometry among premenarcheal girls. The placebo group was significantly different (P ¼ 0.04) from the others in Hb concentration. The iron-only group had the lowest SF concentration (13.5 mg/l), which was different from that of the combine-supplemented group (20.9 mg/l; P ¼ 0.05). Zincsupplemented groups had lower SZn concentration over the other two groups, although not significantly. There was no treatment effect on weight of premenarcheal females with micronutrient supplementation (Table 3) but the zinc-only supplemented group had significant improvement in height (mean difference of 1.7 cm, P ¼ 0.04). Further, zinc-supplemented groups had significant improvements in WAZ and HAZ (P ¼ 0.03). Iron-supplemented groups (iron only and combine-supplemented groups) had significant improvements in Hb (P ¼ 0.05) and SF (P ¼ 0.03) concentrations, but the difference between these two groups was not significant (Table 4). Supplemented groups had similar SZn concentrations.

Figure 2 Mean change in Hb, SF and SZn in interventional groups x Mean change in Hb between anaemic and non-anaemic children in iron-only and zinc-only groups were significantly different (Po0.05). 0 Mean change in SF between iron-sufficient and iron-deficient children in zinc-only group was significantly defferent (Po0.05). Mean change in SZn between zinc-sufficient and zinc-deficient children in 1iron-only (Po0.05), 2zinc-only and combine-supplemented groups (Po0.001) was significantly different. Hb, haemoglobin; SF, serum ferrittin; SZn, serum zinc.

Discussion Micronutrient deficiencies were common in the population studied. Almost half the subjects had either low Hb, or iron stores (SF), or SZn concentrations, and more than 30% had low concentrations of more than one index. This finding agrees with that reported by Lanerolle and Atukorala (1998) that 59% of their study population had depleted iron stores (SFp12 mg/l). There have been no reports on zinc status among Sri Lankan adolescents until we undertook this study. European Journal of Clinical Nutrition

The efficacy of micronutrient supplementation M Hettiarachchi et al

862 Table 3 Anthropometric indices according to sex and maturity with interventiona Parameter

Iron

Zinc

Combine

Placebo

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

Baseline

6 Months

80 82 40

76 78 39

86 73 54

86 64 51

77 66 73

75 52 72

84 56 50

84 53 44

Weight (kg) Male Female- post Female- pre

36.2 (7.9) 40.1 (5.4) 34.5 (8.5)

36.6 (7.8) 40.5 (5.4) 35.6 (8.6)

36.2 (8.6) 39.8 (7.4) 35.0 (8.7)

37.3 (8.7) 40.9 (7.4) 36.3 (8.7)

36.4 (8.6) 41.4 (9.4) 36.2 (7.1)

36.9 (8.5) 42.0 (9.3) 37.5 (7.1)

37.2 (10.6) 39.0 (6.5) 35.5 (7.8)

37.4 (10.5) 39.2 (6.4) 36.0 (7.6)

Height (cm) Male Female- post Female- pre

150.5 (10.8) 151.6 (5.5) 147.1 (8.0)

150.9 (10.7) 152.0 (5.4) 148.0 (8.0)

150.9 (11.4) 151.2 (6.0) 146.1 (9.1)

152.0 (11.3)b 152.2 (5.8) 147.8 (8.7)b

149.0 (9.0) 152.4 (6.4) 147.3 (7.7)

149.5 (9.0) 152.9 (6.4) 148.4 (7.6)

152.4 (10.7) 150.8 (6.0) 148.5 (9.2)

152.7 (10.7) 151.2 (6.0) 149.2 (8.3)

BMI Male Female- post Female- pre

15.8 (1.9) 17.4 (2.6) 15.7 (2.6)

15.9 (1.9) 17.5 (1.9) 16.0 (2.6)

15.7 (2.0) 17.3 (2.6) 16.2 (2.7)

15.9 (2.0) 17.6 (2.6) 16.4 (2.6)

16.2 (2.3) 17.7 (3.2) 16.6 (2.2)

16.3 (2.3) 17.8 (3.2) 16.9 (2.1)

15.8 (2.8) 17.1 (2.2) 15.9 (2.2)

15.8 (2.8) 17.1 (2.2) 16.0 (2.1)

WAZ Male Female- post Female- pre

1.75 1.63 1.59

1.80 1.66 1.60

1.67 1.54 1.23

1.70 1.56 1.24c

1.57 1.19 1.09

1.64 1.233 1.083

1.61 1.58 1.60

1.82 1.72 1.77

HAZ Male Female- post Female- pre

1.26 1.48 1.36

1.23 1.45 1.34

1.23 1.37 1.06

1.25 1.34d 1.07d

1.27 1.06 0.99

1.33 1.03d 1.01d

1.02 1.35 1.60

1.27 1.44 1.77

n Male Female-post Female-pre

Abbreviations: BMI, body mass index; HAZ, height-for-age z score; WAZ, weight-for-age z score. a Results are expressed as mean (s.d.) for weight, height and BMI; WAZ and HAZ are expressed as mean only. There was no difference in each anthropometric parameter at baseline in each category. b Males and premenarcheal females in zinc-only group had significant height gain with intervention (repeated measures ANOVA; Po0.05) c Premenarcheal females of zinc-only (P ¼ 0.01) and combine-supplemented groups (P ¼ 0.02) and post-menarcheal females of combine supplemented (P ¼ 0.01) had significant improvement in WAZ (repeated measures ANOVA). d Premenarcheal females of zinc-only (P ¼ 0.01) and combine-supplemented groups (P ¼ 0.02) and post-menarcheal females of zinc-only (P ¼ 0.03) and combinesupplemented (P ¼ 0.03) had significant improvement in HAZ (repeated measures ANOVA).

The supplement used in the present study had a ratio of iron (mg) to zinc (mg) of 3.6:1. In a metabolic balance study among infants by Haschke et al. (1986), iron to zinc ratios of 5.4:1 and 1.3:1 demonstrated no significant effect on zinc absorption (zinc absorption 15.6 and 20.4%, respectively). Sandstrom et al. (1985) found that increasing molar ratios of iron to zinc from 1:1 to 2.5:1 did not affect absorption of zinc from water but a ratio of 25:1 has been found decreasing. They did not observe an inhibitory effect on zinc absorption when iron and zinc were given with a meal of rice and meat sauce.

Effect on anthropometry The period of supplementation in our study may have been too short to show a significant improvement in growth of children and no reduction in type of undernutrition (data not shown) with intervention. However, in the zincEuropean Journal of Clinical Nutrition

supplemented groups (zinc and combined) a significant improvement (Po0.001) in both weight (mean improvement of 0.94 kg) and gain in height (mean improvement of 0.97 cm) over the other two non-zinc-supplemented (irononly and placebo) groups (0.38 kg in weight and 0.44 cm in height, respectively) was seen. There was no difference in the weight/height gain between the iron-supplemented (iron and combined) groups. Post-menarcheal females had greater improvement in weight and height but zinc-supplemented males showed an improvement in WAZ during the intervention. It is possible to comment that zinc supplementation may show a positive effect on growth of adolescent children if the sample size was larger. Castillo-Dura´n et al. (1994) have reported an increased growth velocity in males with 10 mg of zinc supplementation for 12 months to pre-adolescent and adolescent children. In a randomized control trial among Zimbabwean school children of 11–17 years of age (Friis et al., 1997), two

The efficacy of micronutrient supplementation M Hettiarachchi et al

863 Table 4 Hb and SZn and SF indices according to sex and maturity with interventiona Parameter

Iron

Zinc

Combine

Placebo

Baseline

Six months

Baseline

Six months

Baseline

Six months

Baseline

Six months

80 82 40

76 84 33

86 73 54

86 64 51

77 66 73

75 52 72

84 56 50

84 53 44

119.7 (1.1) 119.0 (1.1) 114.1 (1.1)

128.2 (1.1) 125.0 (1.1)b 123.7 (1.1)

117.5 (1.1) 115.0 (1.1) 115.8 (1.1)

128.6 (1.1) 126.9 (1.1)b 126.1 (1.1)b

125.0 (1.1) 116.3 (1.1) 121.9 (1.2)

123.8 (1.1) 114.3 (1.2) 119.2 (1.1)

n Male Female- post Female- pre Hb Male Female- post Female- pre

114.0 (1.1) 107.8 (1.2) 113.1 (1.1)

131.2 (1.1) 129.0 (1.1) 130.2 (1.1)

b b

SF Male Female- post Female- pre

17.3 (2.1) 16.1 (2.3) 13.5 (2.3)

59.6 (1.5) 52.2 (1.5)c 51.8 (1.6)c

36.9 (1.8) 27.3 (1.9) 27.7 (1.8)

40.0 (1.6) 31.2 (1.8) 30.8 (1.5)

26.2 (1.9) 20.7 (1.9) 20.9 (1.9)

61.1 (1.5) 52.1 (1.5)c 51.8 (1.5)c

37.6 (1.8) 31.0 (1.9) 33.3 (1.9)

36.2 (1.9) 32.9 (1.7) 31.8 (1.9)

SZn Male Female- post Female- pre

10.5 (1.4) 9.9 (1.5) 9.4 (1.5)

10.9 (1.3) 10.6 (1.3) 11.0 (1.2)

7.8 (1.5) 6.4 (1.6) 7.8 (1.5)

12.5 (1.3)d 11.6 (1.2)d 11.7 (1.2)

8.2 (1.5) 7.6 (1.5) 8.0 (1.5)

12.3 (1.2)d 11.7 (1.3) 11.9 (1.2)

10.1 (1.4) 10.4 (1.3) 10.1 (1.3)

11.0 (1.2) 10.5 (1.2) 10.7 (1.2)

Abbreviations: Hb, haemoglobin; SF, serum ferritin; SZn, serum zinc. a Results are expressed as mean (s.d.). The differences in each category at baseline are described in text. b Post-menarcheal females in iron-only, zinc-only and combined-supplemented groups (Po0.001) and premenarcheal females of iron-only and combinesupplemented groups (Po0.001) had significant improvement in Hb with intervention (repeated measures ANOVA). c Both female categories of iron-only (P ¼ 0.02) and combine-supplemented (P ¼ 0.03) groups had significant improvements in SF concentration (repeated measures ANOVA). d Males of zinc-only (P ¼ 0.05) and combine-supplemented (P ¼ 0.05) and premenarcheal females of zinc-only (P ¼ 0.03) groups had significant improvement in SZn concentration (repeated measures ANOVA).

regimen of zinc (30 and 50 mg) supplementation for a period of 12 months, resulted in weight gain and WAZ improvement after 3 months of supplementation but the effects have disappeared thereafter. In a study among children of 4–11 years with short stature, iron and zinc supplementation for a period of 1 year improved growth rates (Perrone et al., 1999). Further, only the subjects whose SF levels were higher than 20 mg/l before supplementation in the zinc-supplemented group showed a similar improvement in growth rate. They concluded that iron þ zinc supplementation could be a reasonable treatment to pre-pubertal children affected by marginal zinc and iron deficiency. A randomized placebo control trial among Iranian children aged 8–11 years, when given zinc (10 mg/day) daily for 6 days per week, for 7 months reported a significant increase in weight and length (Ebrahimi et al., 2006). A recent study in India (Sarma et al., 2006) also has shown a significant increase in mean increments of HAZ and HAZ among school children aged 6–16 years when supplemented with a micronutrient fortified beverage (iron, zinc, calcium, folic acid, vitamins and iodine are the key ingredients) for a period of 14 months.

Effect on deficiency prevalence and haematology Results of the present study highlight interesting findings about the prevalence of subclinical micronutrient deficiency

in otherwise healthy school children who live in semi-urban areas of Sri Lanka. Improving iron and zinc nutriture and preventing iron and zinc deficiency in adolescent children has a direct benefit on the well-being of this population. When the iron-only and combined groups were taken together, the prevalence of anaemia was reduced from 65 to 15%, whereas in non-iron-supplemented groups the reduction was less (from 56 to 34%). A similar effect was shown in a supplementation study with iron-fortified whey drink among children and adolescents in Brazil (Miglioranza et al., 2003), where the prevalence of anaemia decreased from 42% at baseline to 26.4% after 6 months (Po0.001) and to 9.6% after 1 year. A marked reduction in the prevalence of iron deficiency in the iron-supplemented groups (by 75% in iron-only and 60% in the combination supplemented) in this study clearly explains the efficacy of supplementation in combating subclinical iron deficiency. The prevalence of zinc deficiency in the zinc-supplemented groups was reduced from over 65 to 25% as a result of intervention. SZn concentrations were increased by 3.9 and 4.3 mmol/l in the two zinc-supplemented groups. A similar increase has been reported in supplementation trials among Vietnamese children (Thu et al., 1999) and in Guatemalan children (Cavan et al., 1993). Although SZn concentration is not a reliable indicator of total body zinc status, the average change seen in the supplemented groups in comparison to the placebo group does suggest that supplementation European Journal of Clinical Nutrition

The efficacy of micronutrient supplementation M Hettiarachchi et al

864 improved zinc status and that zinc and combined supplementation have shown the same effect. Although an elevation of SZn is evident in the iron alone and placebo groups, the magnitude of the increase was markedly less than that of in the zinc-supplemented groups.

Iron–zinc interactions Whittaker (1998) reported that the increase in SF and SZn was somewhat lower when supplemented in combination probably due to the interactive effects of iron and zinc during absorption. Yadrick et al. (1989) reported that supplementation with zinc resulted in a decrease of SF by 23%, using iron and zinc supplements in equal (50 mg/day) amounts among females 25–40 years of age after 10 weeks of supplementation. Our data do not show such a decline in SF even in the zinc-only group possibly due to the smaller dose used and longer duration of supplementation. To study any interactions between iron and zinc, we used the baseline deficiency status of these micronutrients and Hb concentrations. Donangelo et al. (2002) observed a 35% decline in plasma ferritin concentration with zinc supplementation for 6 weeks (22 mg of zinc per day) in women with marginal iron status. In this study, a 10% decline of SF was observed in ironsufficient children with zinc supplementation as compared to a 25% improvement in those with marginal or low iron stores at baseline. Therefore, our data suggest that when the dietary reference intake of zinc (14 mg) was supplemented for 6 months, there may be a reduction in iron stores of children having sufficient stores. Historically, the antagonisms reported in supplementation studies have been attributed to competition between iron and zinc for transport by the divalent metal transporter-1 (DMT1) found in enterocytes of the small intestine (Kordas and Stoltzfus, 2004). However, Yamaji et al. (2001) examined the absorption of iron in the presence of excess zinc and the absorption of zinc in the presence of excess iron. Iron loading decreased DMT1 expression and reduced subsequent iron uptake but did not affect zinc absorption. Conversely, cell loading with zinc increased DMT1 mRNA levels and expression, increased iron absorption, increased ironregulated mRNA (IREG1) expression, but did not change zinc absorption. Our findings suggest that iron significantly inhibited the uptake of zinc only when children were not zinc-deficient: iron supplementation to SZn sufficient children had resulted 9% drop in zinc status while zincdeficient children had a 25% improvement in SZn concentration. It is also possible that the decline in iron status among iron-sufficient children in the zinc-supplemented groups reflects changes in cell metabolism induced by an improvement in zinc status. Zinc is required for gene expression, protein synthesis and immune function (MacDonald, 2000). Supplementation with zinc may suppress subclinical infection or inflammation in these children, which may cause a European Journal of Clinical Nutrition

decrease in acute-phase proteins, including SF. However, other acute-phase proteins such as C-reactive protein were not measured in this study and therefore this theory cannot be verified. The present investigation determined in a group of adolescent school children whether combined-iron and -zinc supplementation would be more effective in correcting deficiency than would iron or zinc-only supplementation. Further, possible interactive effects on sex and with puberty of females were also evaluated. The concordant biochemical response in all four supplemented groups suggested good adherence to the protocol and the efficacy of the supplementation. As the supplemented zinc dose was nearly equal to the RDA of zinc, this outcome confirms that it is possible to improve zinc status in a population with a marginal zinc status by implementing a dietary intervention programme. In addition, severe and moderate forms of anaemia were also successfully treated in children who received iron supplementation. Furthermore, the high initial prevalence of low zinc and iron stores were improved significantly with micronutrient supplementation during the study. This study, therefore, suggests that iron supplementation alone is more effective than if combined with zinc in improving Hb concentrations in anaemic children at baseline. Further, zinc alone is more effective than in combination with iron in improving SZn concentrations in zinc-deficient children at baseline. However, iron stores were improved in iron-sufficient children with iron alone or in combination with zinc supplementation more than in iron-deficient children at baseline. In conclusion, the results of our study suggest possible systemic interactions between zinc and iron, depending on the baseline deficiency status, after 6 months of supplementation. Zinc alone or in combination with iron did not show a significant improvement in growth in adolescents and, long-term supplementation studies are required to define the response to zinc supplementation in this specific-age group. Our data suggest, however, that micronutrient supplementation can be an efficacious arm of a multifaceted approach to the problem of micronutrient deficiencies in developing countries.

Acknowledgements We thank Professor Sarath Lekamwasam for the valuable comments made on the statistical analysis and the Astron Limited, Colombo for manufacturing of mineral capsules. The study was funded by the International Atomic Energy Agency (IAEA-SRL-11958).

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